The purpose of this research is to provide a flexible prenatal genetic testing product that can be expanded to detect any inheritable trait as early as 5, and up to 20, weeks of gestation, from a safe, noninvasive Pap smear. Published studies, as well as our own experience, show that perinatal Pap collections using a cervical cytobrush pose no risk to mother or fetus, and captures trophoblast cells that migrate from the placenta into the reproductive tract. Trophoblast retrieval and isolation from the cervix (TRIC) efficiently isolates hundreds of trophoblast cells without limitations due to early gestational age, maternal obesity, or uteroplacental insufficiency disorders. In our report published in Science Translational Medicine, we isolated sufficient genomic DNA from intact fetal trophoblast cells obtained by TRIC at 5-19 weeks of gestation (n=20) to definitively distinguish maternal and fetal DNA by targeted next-generation sequencing (NGS) of short terminal repeats (STRs) and single nucleotide polymorphisms (SNPs). Compared to massively parallel sequencing of cell-free fetal DNA from maternal serum, which has a fetal fraction of only 4-10% at week 10 of gestation, the complete genome obtained by TRIC has a fetal fraction of 85-100%, and provides nucleotide-specific haplotyping. In our Phase I award, we developed this technology for prenatal genotyping of single gene disorders located on the hemoglobin B (HBB) gene, using custom multiplex PCR amplification of SNPs, STRs and loci across HBB for NGS. We successfully haplotyped the locus for the sickle cell disease (SCD) point mutation and genotyped the remainder of the HBB exome, which includes alternate SCD loci, beta thalassemias and anemias. In Phase II, we will expand the test to include HBA1 and HBA1 genes (alpha thalassemias) to provide a comprehensive hemoglobinopathy screen. Phase I studies revealed that genotyping was consistently accurate, unless cervical specimen collection was suboptimal and <40 trophoblast cells were isolated. Commercialization will require operators to obtain adequate specimens. We will accomplish six milestones towards commercialization of this test: 1. Incorporate the HBA1 and HBA2 loci into a comprehensive hemoglobinopathy test. 2. Optimize success rates through operator training and innovations in collection device designs to increase cervical cell recoveries. 3. Innovate a novel alternative method to cytobrush-based cervical collection. 4. Automate the TRIC processing pipeline for high throughput cell isolation, DNA purification and NGS. 5. Establish sample quality assessment tools powered by artificial intelligence and machine learning. 6. Perform a clinical validation trial to assess test performance. With an estimated annual market potential over $284 million for prenatal hemoglobinopathies testing, the envisioned technology will fill an existing gap in clinical diagnostics and outcompete existing invasive prenatal testing. Our initial commercial product will to enable management of high-risk pregnancies and provide valuable information to physicians and patients in the process of establishing families. Specifically, this initial product will benefit pregnancies at risk of having a child with SCA or other hemoglobinopathies such as thalassemia and anemia.